Magnetic resonance imaging (MRI) is a powerful diagnostic method that yields three-dimensional images of body tissues in vivo. The tissue features obtained are the result of variations in the distribution of water in these tissues. MRI contrast agents administered prior to imaging alter the relaxation times of protons in their vicinity enhancing specific features of an image. MRI contrast agents improve the sensitivity and utility of MRI diagnostics.
The use of contrast agents for MRI in the clinical setting has become a routine standard of practice for the enhancement in resolution and tissue specificity of medical MRI images. Paramagnetic metal chelates, such as Gd(III)-diethylenetriaminepentaacetic acid (Gd(III)-DTPA) (Magnevist), Gd(III)-N,N′,N′,N″,N′″-tetracarboxymethyl-1,4,7,10-tetraaza cyclododecane (Gd(III)-DOTA), and their analogs have proven to increase the relaxation rate of surrounding protons and have been widely used as MRI contrast agents.
The thermodynamic stability of gadolinium complexes are strongly pH dependent, and while the pH in vivo is not highly variable, the current manufacturing methods yield compositions of gadolinium complexes that vary considerably in pH. Reduced thermodynamic stability can result in the release of toxic Gd(III) ion from the ligand, and may be linked to nephrogenic systemic fibrosis. While the formation of the Gd(III) ion occurs due to manufacturing variations in product pH, and this product pH eventually equilibrates to in vivo pH when injected, the dilution due to injection is sufficiently rapid compared to pH equilibration to separate Gd(III) ion and the ligand (for example, pentetic acid), such that when favorable pH is reached, the metal ion and ligand are sufficiently separated that they do not recombine as a conjugate of ligand and gadolinium.
Consequently, medical contrast agents with a large pH range in the product specification present a safety concern regarding product stability and the potential for formation the release toxic Gd(III) from the complex. The large pH range in the drug product is linked to the use of solvents in the drug purification process, which tends to remove ligand in an unpredictable fashion.
The synthetic methods attempted in the past to prepare paramagnetic metal chelates have one or more drawbacks such as the use of large excess of ligand to reduce free Gd(III) ion; or the need to carry out extensive solvent purification of product due to impurities in the original reagents. Ironically, the biocompatible solvents used to purify the drug product can complex with the impurities they are meant to remove. If pure ingredients are used initially, the need for solvent purification is removed. Nevertheless, solvents are still needed because the gadolinium complex must be precipitated in an anhydrous state in order to formulate the drug product at the therapeutic potency.
The sequence of complexing Gd(III) and the ligand in water, drying, and then reformulating in water is a multi-step process that results in dramatic shifts in the delicate balance between the gadolinium ion and the ligand. Ultimately, this multi-step process is responsible for solvent complexed impurities, shifts in pH and gadolinium-ligand balance. As a consequence, it has become standard in the industry to allow large ranges of pH and meglumine content.
In particular, a significant excess of ligand, for example pentetic acid, is intentionally formulated in the current MRI contrast agent Magnevist®. In Magnevist, the formation of Gd(III) ion is reduced in the presence of excess pentetic acid. The formation of the Gd(III) ion is largely the result of variation in the thermodynamic stability of the macromolecular conjugate of pentetic acid ligand and gadolinium in the presence of solvent.
The shortening of proton relaxation times by gadolinium is mediated by dipole-dipole interactions between the unpaired valence electrons of gadolinium and adjacent water protons. The magnitude of gadolinium magnetic dipole interaction drops off very rapidly as a function of its distance from these protons (as the sixth power of the radius). Consequently, the only protons which are relaxed efficiently are those able to enter the gadolinium metal.
The protons can enter the first or second coordination spheres of the gadolinium metal and metal complex. In coordination chemistry, metal ions are described as consisting of two concentric coordination spheres. The first coordination sphere refers to a central atom or ion (in this case gadolinium). The second coordination sphere can consist of ions (especially in charged complexes), molecules (especially those that hydrogen bond to ligands in the first coordination sphere) and portions of a ligand backbone. Compared to the first coordination sphere, the second coordination sphere has a less direct influence on the reactivity and chemical properties of the metal complex. Nonetheless, the second coordination sphere is relevant to understanding reactions of the metal complex, including the mechanisms of ligand exchange and catalysis.
The protons enter the first or second coordination spheres of the gadolinium metal complex during the interval between an rf pulse and a signal detection. This interval ranges in duration from 105 to 106 protons/second (Brown (1985) Mag. Res. Imag. V 3, p 3).
Gadolinium has seven unpaired valence electrons in its 4f orbital and consequently has the largest paramagnetic dipole (7.9 Bohr magnetons) and exhibits the greatest paramagnetic relaxivity of any element (Runge et al. (1983) Am. J. Radiol V 141, p 1209 and Weinman et al. (1984) Am. J. Radiol V 142, p 619). Consequently, gadolinium has the highest potential of any element for enhancing magnetic resonance images.
In order to take advantage of the large paramagnetic dipole of gadolinium one must recognize the toxicity of free gadolinium metal ion (Gd(III)) in vivo. Thus, the use of gadolinium metal in vivo, for example gadolinium chloride or gadolinium oxide, is not safe and a water-soluble chelate of gadolinium must be used. While a water soluble chelated gadolinium-based contrast agent is safer to inject in patients, the toxicity issues are not entirely solved. Latent toxicity is in part the result of precipitation of the gadolinium that can occur at body pH as gadolinium hydroxide.
However, Gd(III) ion, even if it does not form a water-insoluble compound, can still be toxic, since the reactivity of Gd(III) is very similar to Ca(II), and Ca(II) is ubiquitous in chemical pathways in the mammalian body.
In order to increase solubility and decrease toxicity, gadolinium has been chemically chelated by small organic molecules. To date, the chelator most satisfactory from the standpoints of general utility, activity, and toxicity is diethylenetriamine pentaacetic acid (DTPA) (Runge et al. (1983) Am. J. Radiol V 141, p 1209 and Weinman et al. (1984) Am. J. Radiol V 142, p 619). The first formulation of this chelate to undergo extensive clinical testing was developed by Schering-Berlex AG according to a patent application filed in West Germany by Gries, Rosenberg and Weinmann (DE-OS 3129906 A1 (1981)). The chelate consists of Gd-DTPA which is pH-neutralized and stabilized with an organic base, N-methyl-D-glucamine (meglumine or methyl meglumine).
A direct relationship exists between the concentration of an X-ray attenuator and its efficacy in contrast enhancement. The relationship between concentration and contrast effect is not linear with respect to MRI contrast agents, where a threshold concentration of the paramagnetic entity is required to affect the proton relaxation rates in a physiologic region that is being imaged. Beyond this threshold concentration, any further increase in concentration results in little improvement in contrast enhancement. Thus, MRI contrast agents are formulated as close as practicable to the threshold concentration to help reduce toxic effects not mitigated by chelation. However, if the gadolinium complex is unstable, then the formulation must be hedged and the chelate concentration made greater than the threshold value.
The ionic radii of the trivalent lanthanide cations range from 1.1A for La(III) to 0.85 Å for Lu(III) while Gd(III), sitting exactly in the center of the lanthanide series, has an ionic radius of 0.99 Å, very nearly equal to that of divalent Ca(II). Gd(III) can compete with Ca(II) in the chemical pathways of biological systems, and this substitution potential results in gadolinium toxicity to organisms. In fact, the trivalent ion of gadolinium binds with much higher affinity than the divalent ion of calcium. When bound to a Ca(II)-binding enzyme, lanthanide ion replacement often alters the kinetics of the biological process catalyzed by that enzyme.
The toxicity of gadolinium has placed emphasis on the stability of the gadolinium-ligand (GdL) complex, since the complex form is significantly less toxic than the metal ion form. The thermodynamic stability of a complex simply describes the concentrations of all species present in solution at equilibrium as given by the following equations:Gd(H2O)+L ⇄GdL(H2O)+7H2OK st=[GdL] [Gd] [L]
where Gd is gadolinium ion, L is the ligand, K is the stability constant, GdL is the gadolinium-ligand complex, [L] is the ligand protonation constant, [GdL] is the thermodynamic stability constant of the complex, and [Gd] is the Gd(III) ion formation constant. When solvent is introduced into the equilibrium equation, the thermodynamic stability constant of the complex is reduced and the Gd(III) ion formation constant increased.
Free gadolinium metal ion has 8 inner-sphere sites for water, and the complex form has only 1 inner-sphere for water. The Gibbs free energy of the equilibrium process between complex and free metal ion will have large favorable entropy toward the complex form due to the release of seven of the eight inner-spheres for water. This entropy contribution is referred to as the “chelate effect”. This chelate effect can be compromised by the presence of solvent, which can form binding spheres with solvent rather than water.
In addition, the gadolinium ion-ligand interaction possesses a large electrostatic component that contributes a favorable enthalpy term. The result is that the overall free energy change becomes quite favorable toward the complex form. For these reasons, the solvated Gd(III) ion forms very stable complexes with ligands having more basic donor atoms. That stability is enhanced by the absence of solvent.
The desirability of maximally basic groups in ligands results in the universal selection of ligands comprised of amines. This consideration also explains why amine groups with amide-containing side-chains are considerably less basic than amine groups with acetate side-chains, for example diethylene triamine pentaacetic acid (DTPA) or pentetic acid.
Higher thermodynamic stability of a complex is expressed by a larger thermodynamic stability constant Kst. It should be appreciated that small differences in the ligand protonation constants can have a significant impact on the thermodynamic stabilities of the resulting GdL complex. Unlike the relatively small variations in the log [L] values for the ligands, the log Kst values for a complex can vary by over 10 orders of magnitude. The stability constant is widely used to compare contrast agents because it reduces comparisons to a single convenient number.
The thermodynamic stability constant describes the equilibrium under conditions where the ligand is entirely deprotonated. At physiological pH values, the ligand will be partially protonated so one can argue that a better way to compare GdL stabilities is to use what are called conditional stability constants, set forth in Table 1.
TABLE 1Thermodynamic and Conditional StabilityConstants for Common Gd ComplexesDTPA-DOTA-Ligand protonation constantsDTPABMADOTA(gly)4Thermodynamic stability constants22.4616.8524.714.54pH 14 (log KGdL)Conditional stability constant at18.414.817.212.7pH 7.4 (log Keff)(Schmitt-Willich H, Brehm M, Ewers C L, Michl G, Mueller-Fahrnow A, Petrov O, et al. Inorg Chem 1999; 38: 1134-44.)
Table 1 compares the stability constant of complexes formed between gadolinium and various ligands at pH 14 (deprotonated, and standard “thermodynamic stability constant”) and the conditional stability constant at pH 7.4. Stronger acid conditions clearly results in lower complex stability.
There are additional ionic competitors besides protons that can affect complex stability. For instance, ions like zinc, copper, and iron form very stable complexes with these ligands, and can at the right activation energy force gadolinium out of the less toxic complex state. At the same time, gadolinium has a high affinity for some contaminants and will leave the complex for phosphate, citrate, and carbonate ions which may be present in solution. The magnitude of the effect of these contaminants is generally determined by the thermodynamic stability constant at product pH.
Transmetallation of Gadolinium Complexes
Magnevist® list on its label a pH range of 6.5-8 pH. Reported impurities of gadolinium oxide, used in the preparation of Magnevist, are usually 99.9% pure based on the presence of rare earth metals only. Thus the presence of iron, which may be the source of the yellow color, is not assessed. Iron-DTPA complex is yellow in color.
Much attention has been paid to the potential of Zn(II) to react with a gadolinium contrast agent and displace the gadolinium. Such exchange of one metal for another is termed transmetallation. Of the commonly encountered metal ion contaminants in chemical compounds, Na(I), K(I), Mg(II), and Ca(II), all form very weak complexes with the chelators used in contrast agents and are thermodynamically disfavored from such transmetallation reactions. The order of affinity of contrast agent chelators for other endogenous ions is Fe(III)>Cu(II)>Zn(II).
The fact that transmetallation of gadolinium complexes results in the formation of a more unstable metal complex implies that the synthesis methodology can impact the magnitude of transmetallation. In particular, if reaction temperatures are kept as low as possible during the complexation process can significantly reduce the incidence of non-gadolinium metal complex formation.
Accordingly, there is a need to improve the safety profile of MRI contrast agents. The present disclosure addresses this need by providing a gadolinium complex formulation for injection, where solvent is absent, and the competitive interaction with ligand eliminated.